A yeast‐based tool for screening mammalian diacylglycerol acyltransferase inhibitors

Abstract Dysregulation of lipid metabolism is associated with obesity and metabolic diseases but there is also increasing evidence of a relationship between lipid body excess and cancer. Lipid body synthesis requires diacylglycerol acyltransferases (DGATs) which catalyze the last step of triacylglycerol synthesis from diacylglycerol and acyl‐coenzyme A. The DGATs and in particular DGAT2, are therefore considered potential therapeutic targets for the control of these pathologies. Here, the murine and the human DGAT2 were overexpressed in the oleaginous yeast Yarrowia lipolytica deleted for all DGAT activities, to evaluate the functionality of the enzymes in this heterologous host and DGAT activity inhibitors. This work provides evidence that mammalian DGATs expressed in Y. lipolytica are a useful tool for screening chemical libraries to identify potential inhibitors or activators of these enzymes of therapeutic interest.


| INTRODUCTION
The diacylglycerol acyltransferase (DGAT) enzymes catalyze the final committed step of the triacylglycerol (TAG) biosynthesis by esterification of a fatty acyl moiety to a diacylglycerol. These neutral lipids are stored in organelles called lipid bodies (LBs) in mammalian adipose tissue but also in most eukaryotic cells and some prokaryotes as energy molecules or membrane synthesis reservoirs. In eukaryotes, TAGs are mainly synthesized by DGAT1 and DGAT2 (two different gene families). DGAT1 and DGAT2 have different roles in TAG synthesis in humans: DGAT1 is highly expressed in the small intestine and has a role in fat absorption while DGAT2 is expressed in liver and adipose tissue and is responsible for the endogenous synthesis of TAG (Cases et al., 1998(Cases et al., , 2001. The dgat1 knockout mice are viable with a minor impact on TAG levels and are resistant to diet-induced obesity (Smith et al., 2000). In contrast, dgat2 knockout mice present severe TAG decrease and die shortly after birth (Stone et al., 2004).
And, TAG excess in tissues is a hallmark of obesity. Therefore, DGATs are considered potential therapeutic inhibition targets for the control of obesity, but also for some diseases related to lipid absorption in the intestine. Moreover, recent studies revealed that high levels of LBs are also associated with breast cancer (Nisticò et al., 2021) as well as with higher tumor aggressiveness and chemotherapy resistance (Tirinato et al., 2017). Interestingly DGAT2 is constitutively activated in various cancers including breast cancer (Hernández-Corbacho & Obeid, 2019). In addition, the importance of DGAT2mediated regulation of TAG metabolism in triple-negative breast cancer has been recently highlighted (Almanza et al., 2022).
Therefore, DGAT2 appears as a new potential therapeutic target in the treatment of breast cancer (Hernández-Corbacho & Obeid, 2019 Being able to express these enzymes in a simple heterologous model would provide an efficient and versatile tool to characterize these enzymes and potential inhibitors. To do so, in this work, the oleaginous yeast Yarrowia lipolytica has been used as a heterologous host. This yeast is particularly valuable in this context. It has been a model for lipid metabolism for decades and has a high enzyme production capacity (Nicaud, 2012). Additionally, it can produce large LBs and it is easy to manipulate thanks to the numerous modern genetic engineering toolboxes now available (Larroude et al., 2018).
In particular, a strain deleted for all the genes coding for enzymes with DGAT activities (Q4) is available (Beopoulos et al., 2012). This strain is not able to form LBs anymore. Previous work has shown that DGAT activity in Y. lipolytica can be easily validated, characterized, and modulated by overexpression approaches in this genetic background, allowing restoration of LB formation and TAG accumulation (Aymé et al., 2015;Gajdoš et al., 2016. Those previous works established the efficiency and versatility of the heterologous expression of DGAT in this particular host.
To determine whether the heterologous constructs could potentially be used as tools to measure the activity of these enzymes and thus useful for screening chemical libraries to identify regulatory molecules, the murine and the human DGAT2 were overexpressed in the above-mentioned Q4 strain, as well as the oleaginous fungus Umbelopsis rhamaniana DGAT2, the first DGAT2 identified and expressed in a heterologous host , and the Y.
lipolytica DGATs for comparison. MmDGAT2 and HsDGAT2 have already been expressed in heterologous systems including insect cells and yeast, but mainly for in vitro activity assays (Cases et al., 2001;Kim et al., 2014;Stone et al., 2006;Turkish et al., 2005;Yen et al., 2005).
The DGAT overexpression in the Q4 chassis strains was therefore first evaluated for the LB restoration phenotype and TAG accumulation to evaluate the capacity to use them as in vivo screening tools for DGAT inhibitor candidate drugs. These strains were then exposed to known inhibitory molecules of mammalian DGAT1 and DGAT2. The results showed that the DGATs are functional in our chassis and that inhibitors conserved their specificities and efficacy. This work provided proof of principle for using these strains as a screening system for libraries of molecules to discover new inhibitors or activators of these enzymes of particular therapeutic interest. and monitored by measuring OD at 600 nm every 20 min for 72 h.
Growth rates (r) were calculated using the Growthcurver R package

| Lipid extraction and quantification
All strains show a complementation phenotype by the formation of LBs (Figure 1). Thus, YlDGAT2 overexpression is the highest as expected, as it is the main DGAT for lipid accumulation in LBs in Y.
lipolytica (Gajdoš et al., 2016), while that of HsDGAT2 is the lowest in the condition tested with small LB formation. Protein sequence alignment shows that all the DGAT2 tested here share the essential YFP and HPHG motifs (Liu et al., 2012), however, there is a very large stretch between these two motifs in YlDGAT2 compared to the other DGAT2 ( Figure A1a)

| Evaluation of DGAT2 inhibitor activity on HsDGAT2
As the HsDGAT2 is functional in Y. lipolytica, we therefore evaluated FP-06424439, a specific inhibitor of DGAT2, for its capacity to inhibit the LBs formation complementation phenotype in yeast. The serial concentration of FP-06424439 was tested against the strain Y7378 overexpressing the Human DGAT2. In these experiments, we increase the C/N ratio in the medium to 30 as it increases the TAG accumulation in Y. lipolytica (Gajdoš et al., 2016) and will consequently improve LB visualization. Accordingly, LBs appear bigger in the Y7378 strain without treatment ( Figure 2). Small inhibition of LB formation can be

| Specificity of DGAT inhibitors on DGATs from different origins
The previous experiment established a minimal concentration that inhibits LB formation without affecting growth for the DGAT2-specific inhibitor PF-06424439. This is also in the range of concentrations for which LBs are reduced in MCF7 breast cancer without affecting cell viability (Nisticò et al., 2021).
Therefore, we tested this inhibitor at the same concentration on the six DGAT overexpressing strains selected in this study to evaluate the DGAT specificity. PF-046020110, a DGAT1-specific inhibitor, was also evaluated as a control. PF-046020110 does not affect the different strains overexpressing DGAT, even YlDGAT1, at the concentration tested ( Figure  No significant growth defects were observed for all strains with any of the compounds ( Figure A2). Quantification of fluorescence was also performed in 96-well plate cultures with a microtiter plate reader for the different strains. Results show the same trends, with a strong reduction of fluorescence observed for the strain Y3137 exposed to PF-06424439 and a less pronounced but significant reduction for Y7378 ( Figure 6). For the latter, the basal level of fluorescence is much lower, in line with fluorescent microscopy observations, which also explains the lower amplitude of reduction. For the other strains, no significant reduction was observed in agreement with fluorescent microscopy observations.
F I G U R E 3 Y7378 (Q4 + HsDGAT2) representative growth curve on microtiter plate with increasing concentration of PF-06424439.
F I G U R E 4 Total lipid content of Y7378 (Q4 + HsDGAT2) exposed to different concentrations of PF-06424439 inhibitor in flask culture, evaluated by gas chromatography. Each concentration of inhibitor was evaluated against the control (0 µg/mL). Asterisks correspond to p-value < 0.05.

ACKNOWLEDGMENTS
We would like to thank Joel Haas for kindly providing us with the MmDGAT2 vectors.

CONFLICT OF INTEREST
None declared.

DATA AVAILABILITY STATEMENT
All data generated or analyzed during this study are included in this published article.

ETHICS STATEMENT
None required.